Abstract: The sarcoplasmic reticulum Ca2+ release channel/ryanodine receptor (RyR), is the main component of the "Ca2+ release unit" of cardiac cells. Other cytosolic accessory proteins include calmodulin, FK506 binding protein and sorcin.. In the lumenal side of the channel, calsequestrin binds Ca2+ and is firmly associated to RyRs. The Ca2+ release unit of cardiac cells is therefore a heterologous system that encompasses the RyR homotetramer and several cytosolic and lumenal proteins. The role of accessory proteins and, consequently, the functional output of a fully- integrated Ca2+ release unit during E-C coupling remain incompletely understood. This proposal will dissect the contribution of each of the accessory proteins of RyRs to intracellular Ca2+ release in the heart. We propose that accessory proteins of RyRs are indispensable elements of the Ca2+ release unit of the heart that allow RyRs to counteract the inherently positive feedback of Ca2+-induced Ca2+ release (CICR). To test this hypothesis, we propose: 1) To define the Ca2+ response of purified ("naked") RyRs. Accessory protein-free RyRs will be reconstituted in lipid bilayers and activated by fast [Ca2+] stimuli that resembles the influx of external Ca2+ through sarcolemmal channels, thus providing an integral outline of the intrinsic response of RyRs to Ca2+. 2) To define the kinetics of Ca2+ response of native RyRs, and of purified RyRs in the presence of accessory proteins. Po-[Ca2+] relationships will be obtained for native (SR-embedded) RyR as above, and compared to those obtained with purified RyR in the selective presence of CaM, FKBP, and sorcin. 3) To determine the effect of phosphorylation/dephosphorylation on the response of the Ca2+ release unit to Ca2+. Phosphorylation of RyRs modulate the Ca2+ release in heart. We will determine the effect of PKA and CaMKII on the kinetics of the Ca2+ response of purified, native RyR, and RyRs regulated by individual accessory proteins. This system of increasing complexity that ranges from a single RyR channel to an ensemble of molecules working in unison will define the kinetic properties and mechanisms of regulation of the "Ca2+ release unit" in heart muscle. Results thus obtained will help resolve the effect of individual components of this unit on the intracellular Ca2+ transient of intact cardiac cells.